Putative Transgenic Shoots Research Articles

A robust genetic transformation protocol to obtain transgenic shoots of Solanum tuberosum L. cultivar ‘Kufri Chipsona 1’

The genetic transformation of plants is an important biotechnological tool used for crop improvement for many decades. The present study was focussed to investigate various factors affecting genetic transformation of potato cultivar 'Kufri Chipsona 1'. It was observed that explants pre-cultured for 2days on MS2 medium (MS medium containing 10µM silver nitrate, 10µM BA, 15µM GA3), injured with a surgical blade and co-cultivated with Agrobacterium tumefaciens strain EHA105 [O.D600 (0.6)] for 2days results in maximum transient β-glucuronidase (GUS) expression. The addition of 100µM acetosyringone in MS2 medium also increased rate of transient GUS expression in both the explants. Clumps of putative transgenic shoots were regenerated using the optimised culture conditions from leaf and internodal explants. The stable integration of T-DNA was established using histochemical staining for GUS and amplification of DNA fragment specific to nptII and uidA genes. Within the clumps, around 67.85% of shoots showed uniform GUS expression in all the tissues and about 32.15% shoots show intermittent GUS expression establishing chimeric nature. Uniform GUS staining of the tissue was used as initial marker of non-chimeric transgenic shoots. Quantitative expression of nptII transgene was found to be directly proportional to uniformity of GUS staining in transgenic shoots. The present investigation indicated that manipulation of culture conditions and the medium composition may help to get transgenic shoots with uniform expression of transgene in all the tissues of potato cultivar 'Kufri Chipsona 1'.

Attempts of genetic transformation in Asparagus officinalis L.

Asparagus (Asparagus officinalis L.) is an economically important crop and it is cultivated for its spears. This crop is a perennial and dioecious monocot. Many are the agronomic traits of interest in the asparagus breeding. We are interested to supplement our breeding research with genomic functional studies. To this end, a reliable and efficient protocol for asparagus transgenic plant regeneration has to be set up. Here we report the preliminary results of a research project aiming to optimize a transformation procedure in asparagus. The possibility to transform and obtain transgenic plants, will also allow us to explore the potential of genome editing for basic and applied research in asparagus. Two different explants were used to transform, cut stems and androgenic calli. Two different Agrobacterium tumefaciens strains were tested (AGL1 and C58 GV2260); each one was co-cultivated singly with the two types of explants in different times of incubation (15, 25 and 60 min) for AGL1 and only 60 min for C58 GV2260. Preliminary data showed greater regeneration capacity of androgenic calli in comparison to the cut stems and this enabled us to obtain four putative transgenic shoots.

Agrobacterium–mediated transformation of cry3ca1 gene into kb1 sweet potato cultivar

Sweet potato Ipomoea batatas (L.) Lam is an important food crop in the world as well as in Vietnam. Despite its many benefits, the production of sweet potato is restricted in many areas of the world by diseases, weed, and, particularly, pests. As an alternative, genetic transformation provides the means for complementing conventional breeding to improve sweet potato to resistant to pest. In this study, shoot tip explants of KB1 sweet potato variety were infected with A. tumefaciens C58 carying pBI101/cry3Ca1 con-struct. The selection were occured on callus producing medium (SM) containing 0.5 g/L picloram, 50 mg/L kanamycin and 500 mg/L cefotaxime. Survival embryogenic calli were then transferred to embryo producing medium (EG2) supplemented with 1.0 mg/L ABA and 1.0 mg/L GA3 after 2–3 weeks. Putative transgenic shoots regenerated on medium (RM) adding 0.5 mg/L kinetin and 1.0 mg/L BAP were rooted on root producing medium (RR). The tentative transgenic lines were proved positively by PCR and finalized by Southern hybridization, and biotest in laboratory. Conclusionly, we obtained 62 tentative transgenic sweet potato lines resistant to kanamycin. Among these lines, five putative transgenic lines were proved positively by Southern hybridization and have one copies of the cry3Ca1 gene. Two of them have the lower degree of infestation by sweet potato weevil (Cylas formicarius) than that of untransformed lines.

Robust genetic transformation of sorghum (Sorghum bicolor L.) using differentiating embryogenic callus induced from immature embryos

BackgroundSorghum (Sorghum bicolor L.) is one of the world’s most important cereal crops grown for multiple applications and has been identified as a potential biofuel crop. Despite several decades of study, sorghum has been widely considered as a recalcitrant major crop for transformation due to accumulation of phenolic compounds, lack of model genotypes, low regeneration frequency and loss of regeneration potential through sub-cultures. Among different explants used for genetic transformation of sorghum, immature embryos are ideal over other explants. However, the continuous supply of quality immature embryos for transformation is labour intensive and expensive. In addition, transformation efficiencies are also influenced by environmental conditions (light and temperature). Despite these challenges, immature embryos remain the predominant choice because of their success rate and also due to non-availability of other dependable explants without compromising the transformation efficiency.ResultsWe report here a robust genetic transformation method for sorghum (Tx430) using differentiating embryogenic calli (DEC) with nodular structures induced from immature embryos and maintained for more than a year without losing regeneration potential on modified MS media. The addition of lipoic acid (LA) to callus induction media along with optimized growth regulators increased callus induction frequency from 61.3 ± 3.2 to 79 ± 6.5% from immature embryos (1.5–2.0 mm in length) isolated 12–15 days after pollination. Similarly, the regeneration efficiency and the number of shoots from DEC tissue was enhanced by LA. The optimized regeneration system in combination with particle bombardment resulted in an average transformation efficiency (TE) of 27.2 or 46.6% based on the selection strategy, 25% to twofold higher TE than published reports in Tx430. Up to 100% putative transgenic shoots were positive for npt-II by PCR and 48% of events had < 3 copies of transgenes as determined by digital droplet PCR. Reproducibility of this method was demonstrated by generating ~ 800 transgenic plants using 10 different gene constructs.ConclusionsThis protocol demonstrates significant improvements in both efficiency and ease of use over existing sorghum transformation methods using PDS, also enables quick hypothesis testing in the production of various high value products in sorghum.

Agrobacterium-Mediated Insect Resistance Gene (cry1Aa) Transfer Studies Pertaining to Antibiotic Sensitivity on Cultured Tissues of Broccoli

ABSTRACTSuccessful plant transformation requires an efficient regeneration protocol and a suitable selection method. A genetically engineered, disarmed Agrobacterium tumefaciens strain containing a binary vector pBin-1Aa with cry1Aa (insect resistance) and npt-II (neomycin phosphotransferase-II) genes was used for genetic transformation studies. Effects of antibiotics kanamycin and cefotaxime were studied to determine the aptness of kanamycin resistance as a selectable marker and for cefotaxime in controlling excessive bacterial growth during genetic transformation studies using cultured hypocotyl, cotyledon, leaf, and petiole tissues of Broccoli (Brassica oleracea L var. italica). Leaf and petiole explants exhibited decreased fresh weight as kanamycin concentration increased, resulting in full or partial inhibition of shoot regeneration. A significant or nonsignificant negative correlation occurred between kanamycin concentration and explant fresh weight over the time. Cefotaxime had little effect on regeneration potential. Growth of Agrobacterium cells was controlled at a concentration of 400 mg·L−1 cefotaxime in explants, and maximum putative transgenic shoot regeneration in explants of hypocotyls (44.88%) and cotyledons (36.29%) was obtained on MS (Murashige and Skoog)-selective shoot regeneration medium. Polymerase chain reaction analysis of genomic DNA using specifically designed primers detected the presence of the cryIAa and npt-II genes in kanamycin-resistant broccoli plantlets. Of five randomly selected putative transgenic shoots, three were positive for presence/integration of cryIAa and npt-II genes during T-DNA transfer and integration into the plant genome. Kanamycin and cefotaxime act as effective selective agents during genetic transformation pertaining to antibiotic sensitivity on cultured tissues of broccoli.

Establishment of an efficient Agrobacterium-mediated genetic transformation system in Pelargonium graveolens: an important aromatic plant

Rose-scented geranium is an important aromatic herb, have eminent for oil. The oil of geranium commercially utilized in the perfumery, cosmetic and aromatherapy industries all over the world. It is also helpful to cure many of the diseases, since it possess antibacterial, antifungal, antioxidant, anti-inflammatory and anticancer activities. However rose scented geranium suffer from several biotic and abiotic stresses, which reduced the yield of oil. So there is need to genetically improve the geranium using biotechnological approaches. The present study demonstrates the establishment of direct regeneration and Agrobacterium tumefaciens (LBA4404) mediated transformation protocol in Pelargonium graveolens (cv. CIM-BIO 171). Different media combinations such as benzyl amino purine (BAP), kinetin, naphthalene acetic acid (NAA), and adenine di-sulphate (ADS) were standardised to induce direct regeneration in P. graveolens. The maximum regeneration frequency i.e. 90.56 ± 1.2% per explant was achieved from petiolar segments in medium containing 2.5 mg/l BAP, 0.1 mg/l NAA, 1 mg/l ADS. However, with the leaf explants only 45.94 ± 2.91% frequency was achieved. In the present study, A. tumefaciens strain LBA4404 was used carrying binary vector pBI121 with the gusA as a reporter gene and neomycin phosphotransferase II (nptII) gene as a plant selectable marker. Parameters like bacterial optical density, infection time, acetosyringone concentration and kanamycin concentration were optimised to achieve maximum transformation frequency (69.5 ± 2.3%).The putative transgenic shoots were subsequently rooted on half strength MS medium and successfully transferred to the greenhouse. The transgenic plants were characterised by gus histochemical assay, PCR analysis (nptII-786 bp and gus A- 1707 bp) and Southern hybridization tests using gusA gene probe. The regeneration as well as transformation protocol will no doubt provide the basis to decipher the insights of metabolic pathways in geranium. Also could be useful for genetic improvement, to make it more tolerant/resistant against biotic and abiotic stresses and ultimately fruitful for Indian farmers in agronomic traits like high biomass, oil content, yield and better quality.

First Report of Agrobacterium tumefaciens mediated genetic transformation of aquatic Rice paddy herb (Limnophila aromatica)

The study presents first report of Agrobacterium mediated genetic transformation in Rice paddy herb (Linmophila aromatica). A. tumefaciens strain C58C1 harboring pBin19 Plasmid containing β-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) gene, under the control of 35S promoter and NOS terminator was used. Shoot tip explants were inoculated for 30 min followed by co-cultivation for 72 h and selected on agar semi solidified MS medium containing 100 mg/l Kanamycin and 1.0 mg/l BA; whereas total number of 78 putative transgenic shoots were obtained. The shoots were rooted on MS medium containing 1.0 mg/l IBA and 100 mg/l Kanamycin where 43 plants survived and rooted. Expression of GUS gene in the putative transgenics was confirmed by histochemical GUS assay. Visible localised gus expression was noted in a few cells and callus tissues of 4 plantlets that were photographed using compound light microscope.

Biolistic transformation of Carrizo citrange (Citrus sinensis Osb.×Poncirus trifoliata L. Raf.).

The development of transgenic citrus plants by the biolistic method. A protocol for the biolistic transformation of epicotyl explants and transgenic shoot regeneration of immature citrange rootstock, cv. Carrizo (Citrus sinensis Osb.×Poncirus trifoliata L. Raf.) and plant regeneration is described. Immature epicotyl explants were bombarded with a vector containing the nptII selectable marker and the gfp reporter. The number of independent, stably transformed tissues/total number of explants, recorded by monitoring GFP fluorescence 4weeks after bombardment was substantial at 18.4%, and some fluorescing tissues regenerated into shoots. Fluorescing GFP, putative transgenic shoots were micro-grafted onto immature Carrizo rootstocks in vitro, confirmed by PCR amplification of nptII and gfp coding regions, followed by secondary grafting onto older rootstocks grown in soil. Southern blot analysis indicated that all the fluorescing shoots were transgenic. Multiple and single copies of nptII integrations were confirmed in five regenerated transgenic lines. There is potential to develop a higher throughput biolistics transformation system by optimizing the tissue culture medium to improve shoot regeneration and narrowing the window for plant sampling. This system will be appropriate for transformation with minimal cassettes.

English

Cassava production in India is drastically affected by cassava mosaic disease (CMD) caused by the Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV). An attempt was done to develop transgenic cassava lines resistant to SLCMV through RNAi vector targeting a conserved 440 bp of 5’ end of SLCMV Rep (AC1) gene which also overlaps with part of AC4 gene, and functions as a viral RNAi suppressor protein. The partial Rep gene of SLCMV was cloned in sense and anti-sense orientations in the RNAi intermediate vector, pHANNIBAL and finally mobilised into binary vector pART27, to construct pSCR1 which contains the kanamycin-resistance gene as a plant selectable marker. In order to use hygromycin as selection agent in cassava genetic transformation, Rep-RNAi gene cassettes of SLCMV was cloned into pCAMBIA1305.2 and the constructs was named pSCR2. Agrobacterium mediated cocultivation of cassava embryogenic calli was done with the developed RNAi constructs using two different explants namely, immature leaf lobes and somatic cotyledons. In total, 48 putative transgenic cassava shoots were regenerated on a regeneration medium containing 30 mgl/l hygromycin, of which 2 putative transgenic plants were transferred for hardening. All the putative transgenic cassava plants were PCR-positive for hph gene and Rep gene indicating integration of transgenes of interest. Key words: RNAi constructs, cocultivation, cassava mosaic disease (CMD).

Novel antibiotics regeneration and genetic transformation with RsMYB1 gene of recalcitrant chrysanthemum cv. Shinma

This research was conducted to develop genetic transformation of the recalcitrant chrysanthemum cv. Shinma by application of appropriate antibiotics and selective agents. Clavamox had the least inhibitory effect on shoot regeneration compared to timentin, carbenicillin, and cefotaxime. Clavamox, at a concentration of 125 mg L−1, was found to be the most suitable for shoot regeneration and production of quality shoots, suppressing the growth of Agrobacterium in explants infected with strains GV3101 or C58C1 for 3 and 4 weeks, respectively. The concentration of phosphinothricin (PPT) was found to be 1.0 mg L−1 for screening of putative transgenic shoots. Moreover, transgenic chrysanthemums were obtained by culturing explants co-cultivated with A. tumefaciens strain GV3101 harboring an anthocyanin regulatory gene RsMYB1 isolated from radish (Raphanus sativus), which was placed under the control of cauliflower mosaic virus promoter (CaMV) 35S and petal-specific promoter InMYB1 isolated from the morning glory (Ipomoea nil), on shoot regeneration medium supplemented with recommended concentration of antibiotic and selective agent. Flow cytometry analysis revealed that there was no variation in ploidy level between transgenic plants and donor plants (non-transformants). To our knowledge, this is the first report of the use of Clavamox and MYB transcription factor for genetic transformation of this chrysanthemum.

Agrobacterium-mediated genetic transformation of Populus deltoides marsh clone G48 with gus and npt-II genes

Agrobacterium-Mediated Genetic Transformation of Populus deltoides Marsh Clone G48 with gus and npt-II Genes The present investigation had been carried out to standardize a protocol for Agrobacterium- mediated gene transfer in Populus deltoides Marsh. Clone G48 using petiole explants. Reproducibility of already standardized regeneration protocol for clone G48 had been evaluated. High frequency shoot regeneration (72%) from petiole explants was obtained on MS medium supplemented with 0.5 mg l-1 BAP, 0.2 mg l-1 IAA and 15 mg l-1 adenine sulphate. Root regeneration (100%) in in vitro developed shoots was obtained on MS medium supplemented with 0.1 mg l-1 IAA. Increasing concentrations of kanamycin (10-50 mg l-1) were given to find out the minimum dose of kanamycin required for the selection of putative transformed cells during genetic transformation. It was observed that a doze of 50 mg l-1 kanamycin inhibited callus formation and shoot regeneration and the explants turned brown and started dieing. This concentration of kanamycin would be the most useful for selection of npt-II gene transformed petiole cells/ tissues. Disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter β-glucuronidase (gus) gene in binary vector pBI 121 along with kanamycin resistance gene (npt-II) was used for genetic transformation experiments. Only the transformed cells/ shoots were able to grow on selective shoot regeneration medium containing 50 mg l-1 kanamycin, whereas control explants did not survive. Regenerated putative transgenic shoots were subjected to transient gene expression analysis using spectrophotometric GUS assay and were found positive. This genetic transformation protocol will provide a platform for genetic manipulation of P. deltoides clone G48 for incorporation of genes governing various silviculturally important traits in future....

English

In the present investigation, explants from Kalancho&euml; blossfeldiana were used for gene transformation. The young leaves were inoculated with Agrobacterium tumefaciens LBA4404 strain with a binary vector plasmid pArtblue containing F3&#39;5&#39;H gene under control of CaMV35S promoter and nptII selectable marker gene. After inoculation, the explants were transferred to the co-cultivation medium. They were then transferred to the selection medium containing kanamycin and were sub-cultured every two weeks. Leaves of the putative transgenic shoots that survived in the selection medium were used in reverse transcription polymerase chain reaction&nbsp;(RT-PCR) analysis to detect gene expression. The RT-PCR analysis showed the presence of 550 bp F3&#39;5&#39;H amplification products and had an expression of F3&#39;5&#39;H gene. Plants with the introduced F3&#39;5&#39;H gene produced totally pale red flowers. Key words: Kalancho&euml; blossfeldiana, Agrobacterium-mediated transformation, young leaf, F3&#39;5&#39;H gene, reverse transcription-polymerase chain reaction&nbsp;(RT-PCR).

Improvement of adventitious organogenesis for regeneration of transgenic plants in blackberry

The introduction of foreign DNA into the plant genome by Agrobacterium tumefaciens is a promising technique of targeted gene transfer which depends on good working regeneration system. The aim of the work was to elaborate the system for efficient adventitious organogenesis and transgenic plant regeneration in Rubus fruticosus L. using explants from mature plants. Regeneration of putative transgenic shoots took place from flag explants cultivated vertically on MS medium with 1 mg l-1 TDZ and 0.02 mg l-1 IBA followed by transfer on MS medium with 1 mg l-1 BAP, 0.02 mg l-1 IBA and 0.1 mg l-1 GA3 supplemented with 10-15 mg l-1 hygromycin after transformation by A. tumefaciens strain LBA 4404 carrying plasmid pCambia 1304. Four putative transgenic plants of cv. 'Cacanska Bestrna' were rooted and acclimatized.

An efficient plant regeneration and Agrobacterium-mediated genetic transformation of Tagetes erecta.

Tagetes erecta, L. an asteraceous plant of industrial and medicinal value, contains important compounds like pyrethrins, thiophenes and lutein, possessing immense potential for insecticidal, nematicidal and nutraceutical activities. Considering the importance and demand for these natural compounds, genetic manipulation of this crop for better productivity of secondary metabolites holds great significance. A rapid and reproducible direct regeneration and genetic transformation system is the prerequisite for genetic manipulation of any crop. This paper elucidates the establishment of an efficient direct regeneration and transformation protocol of T. erecta using Agrobacterium tumefaciens. Investigation of the effects of different types of explants (Hypocotyls, cotyledonary leaves, rachis and leaf sections) and different BAP and GA3 combinations on the regeneration frequency of T. erecta suggested that the best regeneration frequency (66%) with an average of 5.08 ± 0.09 shoot buds/explant was observed from hypocotyl explants cultured on media containing 1.5mg/l BAP and 5mg/l GA3. The transformation protocol was established using A. tumefaciens strain LBA4404, containing the binary vector pBI121, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Various parameters like optimization of kanamycin concentration (200mg/l) for selection, standardization of cocultivation time (45min) and acetosyringone concentration (150μM) for obtaining higher transformation frequency were established using hypocotyl explants. The selected putative transgenic shoots were subsequently rooted on the Murashige and Skoog medium and transferred to the green house successfully. The plants were characterised by analysing the gus expression, amplification of 600bp npt II fragment and Southern blot hybridization using the PCR-amplified gusA fragment as probe. The standardised protocol established during the study will open new vistas for genetic manipulation and introduction of desired genes for genetic improvement of T. erecta.

Strawberry (Fragaria × ananassa).

Genetic transformation in strawberry (Fragaria spp.) can be achieved by using the Agrobacterium-mediated procedure on leaves from in vitro proliferated shoots. Regardless of the sufficient regeneration levels achieved from leaf explants of some commercial strawberry genotypes, the regeneration of transformed strawberry plants remains difficult and seems to be strongly genotype dependent. In fact, the main factors that play an important role in the success of strawberry genetic transformation are the availability of an efficient regeneration protocol and of an appropriate selection procedure of the putative transgenic shoots. The strawberry genetic transformation protocol herein described relates to three genotypes resulted from our experience with the highest regeneration and transformation efficiency. The study includes two octoploid Fragaria × ananassa cultivars, Sveva and Calypso, and a diploid F. vesca cultivar (Alpina W.O.). All the different steps related to the leaf tissue Agrobacterium infection, coculture, and selection of regenerating adventitious shoots, as well as the following identification of selected lines able to proliferate and root on the selective agent (kanamycin), will be described.

Agrobacterium-Mediated Transformation of Pomegranate (Punica granatum L.) ‘Yousef Khani’ Using the gus Reporter Gene

In this study, an efficient Agrobacterium-mediated transformation method was developed forpomegranate (Punica granatum L.), a difficult-to-transform plant. In vitro shoot segments wereinoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vectorpBI121 carrying the neomycin phosphotransferase (nptII) gene as a selectable marker and β-glucuronidase (gus) gene as a reporter. After 28 d in WPM selection medium containing 50 mgL-1 kanamycin, 59 new shoots proliferated. gus analysis was performed on these putativetransgenic shoots, of which 32 stained positive. Positive staining shoots were cut and culturedin selection medium for 2 subsequent subcultures until final gus analysis. After three months ofthe selection period, 6 putative transgenic shoots were obtained. Presence of the gus and nptIIgenes was confirmed by polymerase chain reaction. Southern blot analysis confirmed that TDNAwas stably integrated into the genome of three out of six PCR-positive plants. Thetransgenic plants were rooted and successfully acclimatized.

Genetic engineering of apple (Malus domestica Borkh.) for resistance to fungal diseases using g2ps1 gene from Gerbera hybrida (Asteraceae)

In the present study, g2ps1 gene from Gerbera hybrida coding for 2-pyrone synthase which contribute for fungal and insect resistance was used. The aim was to work out an efficient approach of genetic transformation for apple cvs. ‘Golden Delicious’, ‘Royal Gala’ and ‘MM111’, ‘M26’ rootstocks for improving their fungal resistance using genetic engineering techniques. Adventitious shoot formation from leaf pieces of apples studied was achieved using middle leaf segments taken from the youngest leaves from in vitro-grown plants.Optimum conditions for ‚direct’ shoot organogenesis resulted in high regeneration efficiency of 0%, 95%, 92%, 94% in the studied apples respectively. Putative transgenic shoots could be obtained on MS media with B5 Vitamins, 5.0 mg l-1 BAP, or 2.0 mg l-1 TDZ with 0.2 mg l-1 NAA in the presence of the selection agent “PPT” at 3.0-5.0 mgl-1. Shoot multiplication of transgenic shoots was achieved on: MS + B5 vitamins + 1.0 mg l-1 BAP + 0.3 mg l-1 IBA, 0.2 mg l-1 GA3+1.0 g/l MES+ 30 g/l sucrose + 7.0 g/l Agar, with the selection agent PPT at 5.0 mg l-1 and were subcultured every 4 weeks in order to get sufficient material to confirm transformation of the putative shoots obtained. Six, seven, one and six transgenic clones of the apples studied respectively have been obtained and confirmed by selection on the media containing the selection agent “PPT” and by PCR analysis using the suitable primers in all clones obtained for the presence of the selection” bar gene (447 bp) and the gene-of- interest “g2PS1” (1244 bp), with transformation efficiency of 0.4%, 0.6%, 0.1% and 0.3% respectively. These transgenic clones were multiplied further in vitro in the presence of the selection agent ‘PPT’ and rooted in vitro. Rooted transgenic plantlets were successfully acclimatized and are being kept under-containment conditions according to the biosafety by-law in Syria to evaluate their performance for fungal resistance .

Regeneration and Agrobacterium-mediated transformation of three economically important strawberry cultivars Kurdistan, Camarosa and Paros

Genetic transformation studies were carried out to standardize a protocol for Agrobacterium-mediated genetic transformation of three economically important strawberry (Fragaria x ananassa Duch) cultivars Kurdistan’, Camarosa’, and ‘Paros’. Shoot regeneration frequency 72, 65 and 30% was obtained on MS (1) basal medium supplemented with 2% glucose and 4 mg/l TDZ for Camarosa, Kurdistan and Paros, respectively. To optimize the concentration of kanamycin for these cultivars, we observed the responses of leaf explants to different concentration of kanamycin (0-100 mg/l) in the media. Our results showed that 75 mg/l was an effective concentration of kanamycin. For genetic transformation, Agrobacterium tumefaciens strain C58C1RifR (pGV2260) harbouring the binary vector pTJK136 containing uidA gene with intron along with kanamycin resistance gene (npt-II) was used. After five days pre-incubation and 72 h co-cultivation, transformed cells (explants) were able to grow on the selective regeneration medium containing 75 mg/l kanamycin and 500 mg/l cefotaxime, while, control explants failed to grow. The regenerated putative transgenic shoots were analyzed by histochemical GUS assay and PCR analysis. Transformation efficiency based on inoculated explants number was 2, 1 and 3% for ‘, Camarosa’, ‘Paros ’, and ‘Kurdistan’, respectively. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of these cultivars with important agronomic genes.

Transgenic lines of melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) expressing antifungal protein and chitinase genes exhibit enhanced resistance to fungal pathogens

The oriental melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) is an important fruit crop in the tropical and subtropical regions. However, oriental melon production is severely decreased by fungal diseases. In this study, antifungal protein (AFP) and chitinase (CHI) fusion genes were introduced into oriental melons to control fungal diseases caused by Rhizoctonia solani and Fusarium oxysporum. Transformation of oriental melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) with Agrobacterium tumefaciens strain LBA4404 containing antifungal protein (AFP) and chitinase (CHI) fusion genes under the control of the cauliflower mosaic virus (CaMV) 35S promoter and neomycin phosphotransferase (nptII) gene as a selectable marker was performed. Cotyledon explants of oriental melon were inoculated by Agrobacterium suspensions with pBI121–AFP–CHI and cultured in a regeneration medium. After regeneration, genomic DNA polymerase chain reaction (PCR) was conducted to confirm the presence of putative transgenic shoots. Southern blot analysis confirmed that the AFP–CHI fusion gene was incorporated into the genomic DNA of the PCR-positive lines. RT-PCR analysis showed that the AFP–CHI fusion gene was expressed in the individual transgenic lines. Western blot analysis revealed the accumulation of CHI protein in leaves. A segregation analysis of the T1 generation confirmed the inheritance of the transgene. Our results demonstrated that the AFP–CHI fusion gene was effective in protecting the transgenic melon plants against fungal disease caused by Rhizoctonia solani and Fusarium oxysporum.

Agrobacterium-mediated transformation of cotyledons of mature seeds of multiple genotypes of sunflower (Helianthus annuus L.)

Lack of a genotype-independent transformation protocol for sunflower is a major bottleneck in improving this important oilseed crop. An efficient Agrobacterium-mediated transformation protocol is described, which was adaptable across a broad range of sunflower genotypes. The improved transformation approach used cotyledons from mature seeds vertically split through the embryo axis. The LBA 4404 Agrobacterium strain was used, which carried pCAMBIA 2301 plasmid containing UidA as the reporter gene and nptII as the selectable marker for transformants on kanamycin. Bacterial titer, cotyledon type, acetosyringone concentration and vacuum application enhanced the transformation efficiency. Wounding, enzyme pretreatment and sonication significantly reduced the transformation frequency. Putative transgenic shoots were obtained through both axillary proliferation and adventitious shoot regeneration. Following two and three cycles of selection on kanamycin for axillary and adventitious shoots, respectively, putative transformed shoots were obtained at an average frequency of 3.0 %. Reporter gene histochemical assay and molecular characterization of primary and T1 transgenic plants revealed stable transgene integration, expression and monogenic inheritance. The standardized procedure was tested on 28 genotypes comprising sets of inbred, maintainer, restorer and hybrid lines. Transformation was successful in all genotypes albeit with variable frequency in all except the hybrid lines wherein it was stable around 4.0 %. The procedure opens possibilities of directly improving any commercial genotypes of sunflower.